High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana.

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.